Speciation of bioaccumulated uranium(VI) by Euglena

DOI 10.1515/ract-2013-2162 | Radiochim. Acta 2014; 102(5): 411–422

Sina Brockmann, Thuro Arnold*, and Gert Bernhard

Speciation of bioaccumulated uranium(VI) by Euglena mutabilis cells obtained by laser fluorescence spectroscopy Abstract: The ability of Euglena mutabilis cells – a unicellular protozoan with a flexible pellicle, which is typically found in acid mine drainage (AMD) environments – to bioaccumulate uranium under acid conditions was studied in batch sorption experiments at pH 3 and 4 using Na2 SO4 and NaClO4 as background media. It was found that axenic cultures of Euglena mutabilis Schmitz were able to bioaccumulate in 5 days 94.9 to 99.2% of uranium from a 1 × 10−5 mol/L uranium solution in perchlorate medium and 95.1 to 95.9% in sodium sulfate medium, respectively. The speciation of uranium in solution and uranium bioaccumulated by Euglena mutabilis cells, were studied by laser induced fluorescence spectroscopy (LIFS). The LIFS investigations showed that the uranium speciation in the NaClO4 systems was dominated by free uranyl(VI) species and that the UO2 SO4 species was dominating in the Na2 SO4 medium. Fluorescence spectra of the bioaccumulated uranium revealed that aqueous uranium binds to carboxylic and/or (organo)phosphate groups located on the euglenid pellicle or inside the Euglena mutabilis cells. Reduced uranium immobilization rates of 0.93 – 1.43 mg uranium per g Euglena mutabilis biomass were observed in similar experiments, using sterile filtrated AMD waters containing, 4.4 × 10−5 mol/L uranium. These lower rates were attributed to competition with other cations for available sorption sites. Additional LIFS measurements, however, showed that the speciation of the bioaccumulated uranium by the Euglena mutabilis cells was found to be identical with the uranium speciation found in the bioaccumulation experiments carried out in Na2 SO4 and NaClO4 media. The results indicate that Euglena mutabilis has the potential to immobilize aqueous uranium under acid condition and thus may be used in future as promising agent for immobilizing uranium in low pH waste water environments. Keywords: Euglena mutabilis, Uranium, Bioaccumulation, Laser-induced fluorescence spectroscopy (LIFS), AMD, Speciation.

|| *Corresponding Author: Thuro Arnold, Helmholtz Zentrum DresdenRossendorf e. V., Institute of Resource Ecology, P.O.Box 510119, D-01314 Dresden, Germany, e-mail: [email protected] Sina Brockmann, Gert Bernhard: Helmholtz Zentrum DresdenRossendorf e. V., Institute of Resource Ecology, P.O.Box 510119, D-01314 Dresden, Germany Sina Brockmann, Gert Bernhard: Technical University Dresden, Radiochemistry, D-01062 Dresden, Germany

1 Introduction Acid waters containing high amounts of toxic metals are observed in mining regions where coal or sulfidic ore bodies were mined. They are described as acid mine drainage (AMD) environments. In-situ leaching of uranium ores with sulfuric acid, e.g. applied in Königstein/Saxony [1] and in some South African gold and uranium mines [2] also creates AMD which is additionally characterized by elevated uranium concentrations. Such AMD environments are often colonized by benthic microorganisms which form thick and stable biofilms and mats [3–5]. Microorganisms that are able to life under these hostile conditions are species of bacteria, archaea, and eukaryotic microorganisms, e.g. protozoa, algae and fungi [6–8]. The influence of microorganisms in biofilms on the migration and retention of toxic metals in contaminated waters with low pH (pH 2–4) was already discussed in literature, but has focused mainly on the influence of bacteria [9, 10]. Thus, there is only limited information available on the immobilization behavior of eukaryotes or microbial biofilms on metal removal in AMD environments [11, 12]. However, biofilm communities, containing large amounts of eukaryotes, may play a significant role in mediating their environment by actively and passively contributing to metal attenuation through various processes of biosorption and via formation of laminated organosedimentary structures [13]. Some representatives of the euglenoids – which contain about 44 free-living genera and more than 800 species [14] – e.g. the unicellular eukaryotic microorganisms Euglena sp. have been often observed in at- 10.1515/ract-2013-2162 Downloaded from PubFactory at 08/02/2016 12:09:31PM via Helmholtz-Zentrum Dresden-Rossendorf Bibliothek

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tached microbial communities in AMD environments. Euglena sp. have been identified in metal-polluted sites with high acidity [15], e.g. in the AMD affected run-off of the “Gessenwiese”-site near Ronneburg/Germany [16]. Studies with Euglena cells in AMD environments were performed [e.g. 17, 18], however, almost all of these studies do not address the bioaccumulation of uranium. The effect of pH, temperature and dissolved solids on the growth of Euglena mutabilis in coal mining AMD-systems were studied [17] and orange crystalline-like structures within the cells were observed and related to sequestered iron precipitates. In another study the interactions of Euglena mutabilis with As and Fe were investigated and it was observed that As and Fe were bioaccumulated in and on Euglena cells [18]. Studies on the interactions of Euglena sp. with uranium are, however, so far very limited. In two studies [19, 20] a significant accumulation of uranium by unspecified Euglena sp. cells thriving in tailings at the Elliot Lake (Ontario) were observed and from this it was concluded that specific algae must be important in the transfer of uranium from the hydrosphere to the sediments. However, there was no information provided concerning the reaction mechanisms to immobilize uranium by the Euglena cells. Trenfield et al. [21] studied the influence of dissolved organic carbon on uranium toxicity to Euglena gracilis at pH 6. Both groups [19, 21], however, did not provide any information on the respective uranium speciation in the surrounding water and associated with Euglena cells. In this study, for the first time the bioaccumulation of uranium by Euglena mutabilis, a eukaryotic organisms often observed in microbial communities forming biofilms and mats in AMD environments [13, 15] was investigated by laser-induced fluorescence spectroscopy (LIFS) at pH 3 and 4. This technique has superior sensitivity for fluorescent metal ions, e.g. uranium(VI), and is also non-invasive in character [22]. It is suitable for direct determination of the uranium speciation at environmentally relevant uranium concentrations. The term speciation refers to the identity of the element, its oxidation state, its physical state, its stoichiometry, and its detailed molecular structure [23]. Metal speciation determines its reactivity with surfaces and ligands. In our study the term bioaccumulation refers to the uptake of metals from the surrounding solution and comprises sorption processes on cell walls as well as metal uptake into cells. The main objective of this work was to spectroscopically study the interactions of living Euglena mutabilis cells with aqueous uranium(VI) and to provide on a molecular scale new information on the speciation of bioaccumulated uranium. For evaluating metal transport processes in uranium-contaminated environments informa-

tion on the molecular-level speciation of uranium are required for better predicting its stability, mobility, toxicity, and potential bioavailability to humans and other organisms [24]. Spectroscopic evidence for bioaccumulated uranium helps to identify dominant mechanisms of the uranium accumulation process and may help to develop new remediation strategies for removing uranium from contaminated AMD waters.

2 Material and methods 2.1 Cell culture and growth conditions An axenic strain of Euglena mutabilis Schmitz (# 12249b; locality: Czech Republic, Bohemia, “Groẞteich near Hirschberg”) was obtained from the Culture Collection of Algae, Göttingen, Germany (SAG = “Sammlung von Algenkulturen University of Göttingen”). Euglena mutabilis cells show an elongated shape and are approximately 70 – 90 𝜇m long and 6 – 7 𝜇m wide. A detailed description of Euglena mutabilis cells is provided elsewhere [25]. The cells were cultivated under sterile air supply in a liquid medium which was stirred constantly. To enable photosynthetic activity, the cells were grown under natural light conditions. The liquid growth medium was prepared following the suggested recipe of the SAG which was slightly modified by eliminating the soil extract and by adding additional trace elements. The detailed composition of the

Table 1: The composition of the liquid growth medium used to cultivate Euglena mutabilis Schmitz. Macro nutrients

g/L

CH3 COONa KNO3 (NH4 )2 HPO4 MgSO4 ⋅7H2 O CaSO4 ⋅2H2 O Meat extract Bacto-tryptone Yeast extract Trace elements ZnSO4 ⋅7H2 O CuSO4 ⋅5H2 O MnSO4 ⋅4H2 O CoSO4 ⋅7H2 O H3 BO3 Na2 MoO4 ⋅2H2 O NiSO4 ⋅6H2 O Na2 SeO4 ⋅10H2 O Na2 WO4 ⋅2H2 O

1 0.2 0.02 0.01 0.04 1 2 2 mg/L 10 1 1 1 0.6 0.5 1 1 0.1

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Sina Brockmann et al., Speciation of bioaccumulated uranium(VI) by Euglena mutabilis cells

liquid growth medium is shown in Table 1. After approximately six weeks the cell cultures had reached a higher density clearly visible by the intensive green color. At that time the cells were harvested by repeated centrifugations (4,500 g at 4 ∘ C for 10 min) in sterile polystyrene vials to obtain a sufficient amount of cell pellet (approximately 1 ml cell pellet in each vial) for the following bioaccumulation experiments. The cells were then resuspended in a defined background electrolyte to wash out residues of the growth medium. Subsequently, the living Euglena cells were again separated by centrifugation and the washing solution was discarded.

2.2 Bioaccumulation experiments Bioaccumulation experiments with living Euglena mutabilis cells and uranium were carried out in NaClO4 (9 g/L; ionic strength = 7.3 × 10−2 mol/L) and Na2 SO4 (3.48 g/L Na2 SO4 ; ionic strength = 7.3 × 10−2 mol/L) as background medium. These background media are necessary to compare the laser-induced fluorescence spectroscopy measurements (see section 2.4) with published reference data. Perchlorate has been previously used as background medium in bioaccumulation experiments with uranium and microorganisms [26–28] since perchlorate does not form complexes with uranium. Since AMD environments contain high sulfate concentrations, Na2 SO4 was also used as background electrolyte to approximate AMD conditions. The prepared cell pellets of living Euglena mutabilis cells were suspended in sterile filtrated solutions of 1 × 10−5 mol/L uranium (added as 1 × 10−2 mol/L uranylnitrate) in NaClO4 or Na2 SO4 media, which were previously adjusted to pH 3 and 4, respectively. No uranium precipitation should occur under the specified experimental conditions [29]. The bioaccumulation experiments were carried out in triplicates for the perchlorate background media and in quadruplicates for the sulfate background media to detect possible discordant values. Such discordant values, however, were not observed. The results of these triplicates and quadruplicates were combined and presented as arithmetic means. From these values the standard deviation was calculated. The reason for carrying out one additional experiment in sulfate background media was to generate more biomass for additional laser-induced fluorescence spectroscopy analyses. In addition, two vials without cells were prepared for each experiment to check for wall sorption effects on the vials. The dry weights in the bioaccumulation experiments were obtained by drying three times 1 ml of cell sus-

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pensions at 105 ∘ C for two days and subsequently weighing the sample. The use of different bioreactors with Euglena cells for the individual batch experiments resulted in slightly different Euglena dry weight/L biomasses. The dry biomass was then determined by forming the arithmetic mean which accounted for 0.82±0.19 g dry weight/L and 0.42 ± 0.12 g dry weight/L for the bioaccumulation experiments using perchlorate or sulfate as background medium, respectively. The cells were in contact with uranium for five days on a rocking shaker under natural light conditions. Then, the supernatant and the cells were separated by centrifugation (4500 g at 4 ∘ C for 10 min) and the solutions were analyzed for uranium by inductively coupled plasma mass spectrometry. An ELAN 9000 type ICP-MS spectrometer (Perkin Elmer SCIEX, Waltham, Massachusetts, USA) was used for the uranium analyses. The analytical error for the ICP-MS measurements for uranium ranges, depending on the respective U-concentration, from 2 to 10%. The amount of uranium immobilized by Euglena mutabilis cells was calculated from the difference between initial and final uranium concentration of the supernatant. The amount of uranium adsorbed on container walls was determined and if necessary considered in the calculation. The initial solutions and the Euglena cell pellets after being 5 days in contact with uranium were investigated by laser-induced fluorescence spectroscopy (LIFS) to obtain information on the present uranium speciation. Initial solutions are referred to as the uranium solutions before they were brought in contact with Euglena mutabilis cells. Additional bioaccumulation experiments were carried out to study the effects of natural uranium containing waters on the uranium biosorption process on Euglena mutabilis cells. In these experiments the Euglena cells were added to sterile filtrated water obtained from a former underground uranium mining site in Königstein (Saxony, Germany), which represents AMD conditions.

2.3 Sampling of natural uranium contaminated water The water sample was collected from a drainage channel (pH = 3.1) in a mine gallery of the uranium mine in Königstein (Saxony, Germany) in March 2010. The location in Königstein and the sampling procedure there was described in more detail elsewhere [1]. The water sample was taken in sterile glass bottles and transported directly after sampling to the laboratory. There, the water was filtrated through a sterile filter (Whatman, cellulose nitrate, 0.2 𝜇m) and subsequently stored in the re- 10.1515/ract-2013-2162 Downloaded from PubFactory at 08/02/2016 12:09:31PM via Helmholtz-Zentrum Dresden-Rossendorf Bibliothek


Table 2: Chemical composition of the AMD water sample from the underground mine in Königstein. Element

𝜇g/L

ae

Element

𝜇g/L

ae

Na Mg Al Si P K Ca Cr Mn Fe Co Ni Cu

20100 12300 16800 15600 < 10 11900 126000 30.0 3840 78300 144 302 15.2

±400 ±370 ±1700 ±1600 bd ±240 ±2500 ±3 ±120 ±1600 ±3 ±20 ±1

Zn As Sr Cd Cs Ba Pb U

6400 18.2 760 57.4 6.32 12.3 279 10400 mg/L 27.8 771 1.40

±320 ±2 ±23 ±2 ±0.1 ±1 ±14 ±210

Cl− SO4 2− TOC

±3 ±80 ±0.1

pH = 3.1 ae: analytical error; bd: below detection limit.

frigerator at 4 ∘ C. The water samples were analyzed for cations by inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS) for Na, K und Ca and graphite furnace atomic absorption spectrometry (GFAAS) for Fe. An ELAN 9000 type ICPMS spectrometer (Perkin Elmer SCIEX, Waltham, Massachusetts, USA), a Perkin Elmer 4100 AAS, and a AAS-6F ZEEnit 600s Graphite furnace AAS with Zeeman background correction (Analytik Jena, Jena, Germany) were used for these analyses. The error of the chemical analysis for AAS analyses is only 1% – 2% and 2% – 10% for ICP-MS measurements. Anions, i.e. chloride and sulfate were determined by ion chromatography (ICsystem 732/733, Metrohm, Filderstadt, Germany). The analytical error for the anion analyses is smaller than 10%. Total organic carbon (TOC) was determined with the HT1300-TOC equipment (Analytik Jena, Jena, Germany). The analytical error for this technique is smaller than 10%. The chemical composition and the pH of the water sample from Königstein are shown in Table 2.

2.4 Laser fluorescence measurements Laser induced fluorescence spectroscopic measurements were carried out using a Nd-YAG laser (Minilite, Continuum) as excitation source and a spectrograph (iHR 550, HORIBA Jobin Yvon) together with an ICCD camera (HORIBA Jobin Yvon) for detecting the fluorescence light. For all measurements in this study an excitation wavelength of 266 nm was used (laser energy of approximately 250 𝜇J). All spectra were recorded from 371.4 nm

to 674.3 nm by accumulating 100 laser pulses using a gate time of 2 𝜇s. The computer software Labspec 5 (HORIBA Jobin Yvon, Edison, NJ, USA) controls the system as well as the recording of the spectra. The fluorescence data obtained were imported into Origin 7.5 (OriginLab Corporation, Northampton, MA, USA) and fitted with the included PeakFit module 4.0. The error in the position of the emission maxima was determined by measuring a standard uranyl solution (5 × 10−5 M, pH 2) prior to each LIFS measurement. From these measurements standard deviations (1𝜎) were calculated and used as device-specific analytical error of LIFS analyses for the six emission maxima of uranyl(VI) containing species. For the LIFS measurements the liquid samples (initial solution and solution after 5 days of contact to the cells) were positioned in a quartz glass cuvette in the laser beam. The Euglena cell pellets were washed to remove any aqueous uranium from the adherent supernatant by resuspending the cells in new uranium free background medium, i.e. sodium sulfate or sodium perchlorate solutions, respectively. Subsequently the cells were centrifuged at higher centrifugal forces (10 000 g/5 min/4 ∘ C) to obtain a compact cell pellet. To measure the immobilized uranium on the Euglena cells, the wet cell pellet was positioned on a sample holder and adjusted so that the laser beam hit the sample. The delay times, i.e. the times between excitation laser peak and opening of the camera, were increased to at least a time of 80 ns to avoid interferences by shortlived autofluorescence contributions caused by cell components or other organic substances in the samples.

3 Results and discussion 3.1 Bioaccumulation of uranium by Euglena cells During the five days of the bioaccumulation experiments with living Euglena cells the intensive green color of the cell suspensions became slightly bleached. The bleaching effect was more distinct in samples with sodium perchlorate background medium. Visualizing the cells under the light microscope (Leica TCS SP2) showed that the cell morphology of many Euglena cells had changed. The cells became more and more roundish and lost their elongated shape. The loss of motility and the rounding of the cells were described as a stress effect related to low pHvalues and/or metal toxicity [30], e.g. uranium [21]. At uranium concentrations > 300 𝜇g/L at pH 6 for Euglena gracilis it was observed that the cells changed shape from a healthy, long spindle form to a “tear-drop“ or cyst-like - 10.1515/ract-2013-2162 Downloaded from PubFactory at 08/02/2016 12:09:31PM via Helmholtz-Zentrum Dresden-Rossendorf Bibliothek


spherical shape [21]. However, hardly any changes in cell morphologies were observed in own bioaccumulation experiments with Euglena mutabilis cells at pH 5 and 6 using uranium concentrations of 1 × 10−5 mol/L uranium, indicating that the low pH was the main stressor. Euglena mutabilis species are often found in natural acidic environments with elevated metal concentrations. They should possess a growth maximum in the pH-range from 1.7 to 4.6 [17]. However, the axenic Euglena mutabilis cells used in this study were not directly isolated from a low pH environment but were obtained from a culture collection. Such a procedure is commonly used to study interactions between microbes and metals. Following the guidelines of the SAG the Euglena cells were cultivated in nearly neutral growth medium. For the bioaccumulation experiments cell pellets of living Euglena mutabilis cells were added to uranium solutions of pH 3 and 4, respectively. The rapid change in geochemical conditions caused stress to the Euglena mutabilis cells so that the above described changes in cell morphology were observed, contrary to expectations since Euglena cells should be well adapted to withstand low pH conditions and high concentrations of metals. Thus, it is assumed that some Euglena mutabilis cells have lost or reduced their ability to withstand low pH conditions and elevated metal concentrations. Similar observations are described elsewhere [15]. The lack of nutrients for 5 days in the vials during the contact time with uranium is another stress parameter for the Euglena mutabilis cells, so that the cell pellets obtained at the end of the bioaccumulation experiments were very likely a combination of living, stressed and dead Euglena cells. The bioaccumulation experiments revealed that during the five days of the experiments in both background media approximately 95% of the initially added uranium Table 3: Results of the model bioaccumulation experiments in NaClO4 and in Na2 SO4 .

Inital pH

3.0 4.0 3.0 4.0

Final pH

Bound Uranium % of initial mg/gEuglena dry weight amount amount sd amount sd

Experiments in NaClO4 (0.82 g dry weight/L) 3.4 99.2 1.1 2.8 4.9 94.9 1.2 2.7 Experiments in Na2 SO4 (0.42 g dry weight/L) 3.1 95.9 0.7 5.4 4.3 95.1 0.1 5.3

0.1 0.1

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415

was bioaccumulated by Euglena mutabilis, as shown in Table 3. The bioaccumulation rates were determined by averaging for all applied experimental conditions the respective three (in case of using NaClO4 as background media) or four (in case of using Na2 SO4 as background media) obtained results. These rates were very similar and therefore the resulting standard deviation was rather small, as shown in Table 3. The respective bioaccumulation values are, depending on the applied biomass, equivalent to approximately 5 mg U/g Euglena dry weight in the experiments with sodium sulfate medium and 2.5 – 3 mg U/g Euglena dry weight in the experiments with sodium perchlorate medium, respectively. These values are approximately 300 to 1000 times higher than values reported [20] for bioconcentrated uranium by Euglena cells which they collected from mine tailing waters in Elliot Lake (Ontario, Canada), which was known for its uranium mining activity in the second half of the twentieth century. Comparing our rates of bioaccumulated uranium with studies on the immobilization of uranium by biomass of eukaryotic organisms we find even higher rates reported for laboratory studies using e.g. the green algae Chlorella vulgaris at pH 3 [26] and with non-living Saragassum biomass at pH 2.6 to 4.0 [31]. Here values of up to 560 mg U/g dry weight for Saragassum biomass and approximately 35 mg U/g dry weight for Chlorella vulgaris, a small spherical alga with only 4 to 10 𝜇m cell diameter, were observed. Because of its small cell diameter the biomass of Chlorella vulgaris provides a much larger surface area in respect to its dry weight in comparison with Euglena cells and thus may be responsible for the observed higher uranium sorption capacities. The algae Chlorella is, however, not suited to sequester metals on their cells under low pH conditions since they are typically encountered in neutral pH conditions and do not survive under AMD conditions. The non-living Saragassum fluitans biomass studied by Yang and Volesky [31] was a pretreated and finegrained powder with a higher specific surface area than the living Euglena cells. Their experiments did not provide information on the uranium immobilization process on or in living microorganisms. Table 3 also shows that the adjusted pH values slightly changed during the length of the 5 days experiments. These pH changes are probably caused by excreted metabolites due to the increased stress on the cells. Such metabolites, e.g. amino acids, polyamine compounds, urea and some sugar compounds, produced by Euglena mutabilis have been recently detected [32].

0.2 0.1

sd: standard deviation.



Fig. 1: Fluorescence spectra of the samples recorded in the Euglena uranium system at pH 3 with perchlorate as background electrolyte.

Fig. 2: Fluorescence spectra of the samples recorded in the Euglena uranium system at pH 3 with sodium sulfate as background electrolyte.

3.2 LIFS investigations To study in more detail the immobilization process of uranium on Euglena pellicles or inside Euglena cells, LIFS investigations were performed to obtain spectroscopic information to identify the coordination environment, i.e. the speciation of immobilized uranium. Spectra of fluorescent uranium species from the initial solutions and from uranium bound by Euglena cells were recorded for the two background solutions and the two pH values, respectively. The emission spectra of uranyl(VI) species may show up to six peak maxima. The recorded emission maxima of the initial solutions at pH 3 in NaClO4 medium match exactly the emission maxima of the uranyl reference solution in perchlorate medium described by Bell and Biggers [33], which

are shown as vertical dashed lines in Figure 1. This indicates that the free uranyl(VI)-ion (UO2 2+ ) is the dominating uranyl species in the initial solutions and thus may interact with Euglena mutabilis cells. The spectra obtained for the Euglena cell pellets in NaClO4 solution, on which 99.2% of the originally present aqueous uranium was immobilized, were in comparison to the initial solution significantly shifted to higher wavelengths, as shown in Figure 1. However, these spectra showed a high background noise so that not all six peaks could be extracted by the fitting procedure. Such low signal-to-noise ratios were especially observed in samples with high concentrations of organic compounds as is the case with Euglena cells pellets. In these samples only 5 or 4 peaks maxima could be identified. The emission bands (especially the 2nd , 3rd and 4th , see Table 4) of uranium immobilized by Euglena cells for the samples at pH 3 in perchlorate medium are shifted by about 6 to 8 nm (depending on the respective emission maxima) to higher wavelengths in comparison to the initial solutions. From this it can be concluded that during the bioaccumulation process by Euglena mutabilis cells a new uranium species, on or in the Euglena cells, has formed. Figure 2 shows the fluorescence spectra obtained for the Euglena uranium system at pH 3 in sodium sulfate background medium. For comparison the emission bands of a uranyl sulfate reference solution [1] are shown as dashed lines. The emission maxima of the initial uranium solutions are in good agreement with reported emission maxima for UO2 SO4 , identified in AMD environments [1] clearly indicating that the Euglena cells are surrounded in our bioaccumulation experiments by a solution which is dominated by the UO2 SO4 species. The fluorescence spectra obtained for the Euglena cells, which were in contact for five days with a uranium containing solution (concentration 1 × 10−5 mol/L uranium) showed, in comparison with the acquired fluorescence spectra obtained for the initial solutions, a slight but distinct shift to higher wavelengths. This shift is outside the analytical error ranges and indicates a change in the coordination environment of the immobilized uranium species, clearly showing that the uranium speciation in solution and on or in the Euglena cells is different. Comparable fluorescence spectra were recorded for the uranium bioaccumulation experiments (uranium immobilized by Euglena cells) in sodium sulfate medium at pH 4. Here the recorded spectra showed similar positions of the fluorescence emission bands and an equal shift to higher wavelengths in comparison to the observed emission maxima of the initial uranium solutions. The spectra are completely comparable with the experiments carried out at pH 3. Thus, the newly formed uranium species on or in the Euglena cells at pH 3 and pH 4 seems - 10.1515/ract-2013-2162 Downloaded from PubFactory at 08/02/2016 12:09:31PM via Helmholtz-Zentrum Dresden-Rossendorf Bibliothek


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Table 4: Comparison of the LIFS-emission bands of the uranyl species of the measured samples and of reference compounds.

Measured Sample

pH 3, NaClO4

pH 3, Na2 SO4

pH 4, Na2 SO4

Delay time [ns] Initial solution

80

Euglena cells

80

Initial solution

80

Euglena cells

80

Initial solution

80

Euglena cells

80

Reference sample

1 mv sd (1𝜎) mv sd (1𝜎) mv sd (1𝜎) mv sd (1𝜎) mv sd (1𝜎) mv sd (1𝜎)

471.1 1.0

477.5 1.0 478.5 1.0 476.5 1.0 477.3 1.0

Source

U-SO4 standard* Uranyl in perchlorate*

[1] [31]

Uranyl phosphoryl complexes U-ATP U-AMP UO2 (H2 PO4 )+ / UO2 (HPO4 ) (pH 3) U(VI) phosphoserine

[27] [27] [28] [32]

Uranyl carboxyl complexes UO2 (malonate) UO2 (malonate)2 2− UO2 (glycine)2 2+ (R-COO)2 -UO2

[33] [33] [34] [35]

Uranium reference substances 477.5 470.1

Emission bands [nm] 2 3 488.9 509.6 0.5 0.7 495.0 516.3 0.5 0.7 493.0 513.7 0.5 0.7 494.9 516.0 0.5 0.7 492.8 513.9 0.5 0.7 495.6 517.3 0.5 0.7 Emission bands [nm]

in 0.01 N HClO4 in 0.9% NaCl 3 in mineral medium [see 36] 4 in 0.1 N NaClO4 ∗ reference substance 2

to be identical. Also, the positions of the recorded emission maxima of the uranium bound by the Euglena cells in Na2 SO4 medium were comparable to the emission maxima observed in similar experiments with sodium perchlo-

5

6

532.2 1.2 539.9 1.2 537.1 1.2 538.9 1.2 537.2 1.2 541.3 1.2

556.7 0.9 564.8 0.9 562.5 0.9 564.6 0.9 562.9 0.9 564.4 0.9

582.1 1.6

590.1 1.6 591.8 1.6 591.3 1.6

492.8 487.8

514.1 509.3

537.5 532.7

563.0 558.1

590.1 585.4

516.5 519 517 517.6

540.2 542 541 540.5

564.6 569 565 565.0

593.8

480.9

495.3 497 494 496.9

477 479 478.7 466.0

494 496 495.3 481.6

515 517 516.7 498.1

540 542 540.6 518.0

564 566 565.0 539.0

594 597 594.4 566.0

539.9

563.5

594.0

537.9

562.9

590.0

540.9 540.8 541.6 537.8 539.9

566.4 566.4 566.3 565.4 565.4

538.3

564.8

543.6

569.3

480.9

Mixtures of carboxylic and organic phosphate model compounds with uranylperchlorate (two species) UO2 (acetate)/UO2 (O-phospho-L[36] 462.8 481.7 496.7 517.1 threonine) (200 : 1) UO2 (acetate)/UO2 (fructose-6[36] 463.2 479.3 494.8 515.1 phosphate) (25 : 1) Uranium bound on/in microorganisms Acidithiobacillus ferrooxidans[27]1 type I 496.1 517.2 UO2 2+ -complex type II 495.4 516.8 type III 496.3 517.5 Bacillus sphaericus 9602 (pH 2.8) [28]2 496.1 517.5 Uranyl (5 𝜇M) on/in Chlorella [36]3 480.5 496.8 517.6 vulgaris pH = 4.4 Uranyl (0.1 mM) on/in Chlorella [36]3 481.7 497.7 516.8 vulgaris pH = 4.4 Sulfolobus acidocaldarius pH = 1.5 [37]4 483.6 498.3 520.3 1

4

590.0

ATP: adenosine-triphosphate AMP: adenosine-monophosphate mv: mean value sd: standard deviation

rate background medium. There are only very small differences between the peak maxima of the respective samples at pH 3 and pH 4 in sulfate medium (less than the standard deviation) and in perchlorate medium at pH 3. Thus the - 10.1515/ract-2013-2162 Downloaded from PubFactory at 08/02/2016 12:09:31PM via Helmholtz-Zentrum Dresden-Rossendorf Bibliothek


same uranyl speciation is found for the bioaccumulation experiments independent of the background electrolyte. However, the observed shifts to higher wavelengths between the dominating uranium species in the initial solution, i.e. the uranyl sulfate ion (UO2 SO4 ), and the immobilized uranium species associated with Euglena cells are smaller and accounted for 1.8 to 4.1 nm for the main emission maxima, i.e. the 2nd , 3rd , 4th and 5th peak. This shift, despite being smaller than in the uranium-EuglenaNaClO4 system, is still significant and is clearly indicating the formation of a newly formed bioaccumulated uranium species in or on Euglena cells. The fluorescence spectra recorded for this immobilized uranium species, located on or in the Euglena cells, were compared in a fingerprinting fashion with available LIFS reference spectra to extract information on its coordination environment. For that purpose, suitable reference uranium compounds with known coordination environment were selected, e.g. comparable systems with microorganisms and uranium, and used for comparison. All positions of the measured fluorescence emission maxima of the Euglena mutabilis uranium systems at pH 3 in perchlorate medium and at pH 3 and 4 in sulfate medium, respectively, together with emission bands of selected uranium(VI) reference compounds used for the interpretation are summarized in Table 4. The comparison with reference compounds of known uranium coordination showed that the emission bands observed in our Euglena mutabilis study are comparable with several reference compounds and are also similar to emission maxima reported for uranium bound by other microorganisms. However, the number of available reference data is still limited and not all uranium emission bands for possible uranium compounds are known. There is a very good agreement with data reported by [27] for U-adenosine-triphosphate (U-ATP), representing uranium bound to (organo)phosphate functional groups, and with fluorescence data reported by [36] for UO2 (glycine)2 2+ , which are exemplary for carboxylic functional groups. Emission values of uranium bound by Acidithiobacillus ferrooxidans eco-type II [27]- bacteria also known to live in AMD environments -ŋ agree also with the measured values of this study. Merroun et al. [27] found a good agreement of their LIFS results of uranium bound by Acidithiobacillus ferrooxidans eco-type II with the values obtained for U-ATP and U-AMP reference compounds, as well as with complexes formed by uranium and B. sphaericus. This immobilization of uranium by Acidithiobacillus ferrooxidans was attributed to the exclusive binding of uranium on organic phosphate groups of phospholipids of the cell membranes and/or to the binding to polyphosphate bodies in

the cells [27, 40]. However, the emission bands of the uranium bound to the Euglena mutabilis cells are also close to values of uranyl immobilized by B. sphaericus [28] and Chlorella vulgaris [38]. Panak et al. [28] found strong evidence for uranium complexation on Bacilli occurring in inner sphere coordination with phosphate groups of the bacterial cell walls. Vogel et al. [38] found that for algal cells, both carboxylic and organic phosphate groups as well as additionally inorganic phosphate groups for dead algal biomass are involved in the binding of uranium. The euglenoid pellicles consist largely of proteins and lesser amounts of lipids and carbohydrates [41]. Glycine, as amino acid, is known as a basic module of many proteins in cells. As discussed elsewhere [42] the metalbinding in algae is caused by proteins within a mol. wt range of 8000–10 000 and these proteins contain large amounts of the amino acids glutamic acid, cysteine and glycine. Polypeptides composed of these three amino acids are also denoted as phytochelatins, which play an important role for detoxification of e.g. metals for higher plants and algae [43]. Furthermore, the tripeptide glutathione – consisting of the three amino acids cysteine, glycine and glutamate – is also a protein which could possibly bind uranium by forming carboxyl complexes. A uranium-binding process by such proteins at or in the Euglena cells is therefore possible. The formation of immobilized uranyl species with glycine related compounds is another possible uranium immobilization reaction as the LIFS-results of this study have indicated. However, it was not possible to clarify if additional uranium related complexes were also formed on or in the cells since many of these possible uranyl(VI) complexes have not yet been studied by LIFS. Additional U(VI) complexes, which could possibly form, e.g. the uranyl carboxyl complex with glutamic acid [37] and the uranyl cysteine species [36], which may be both relevant in this study, could not be observed with laser fluorescence techniques because they do not show fluorescence properties at room temperature. Also, LIFS measurements performed under cryogenic conditions would not be a suitable tool to resolve these questions, since such measurements carried out in biological systems, e.g. the Euglena system with high concentration of organic compounds would lead to a prolongation of the fluorescence signals of both possibly present fluorescent metal species with a very short fluorescence signal, as well as the prolongation of organic autofluorescence signals, e.g. the chlorophyll fluorescence. Since these organics compounds are in terms of quantity dominant, they would cover up small contributions of possibly present fluorescent metal species with very short fluorescence lifetimes. - 10.1515/ract-2013-2162 Downloaded from PubFactory at 08/02/2016 12:09:31PM via Helmholtz-Zentrum Dresden-Rossendorf Bibliothek


The formation of immobilized uranyl species related to (organo)phosphate groups, found in ATP or ATP related compounds in the uranium Euglena system, are also possible. ATP is used as energy source for basic and specific energy consuming processes of all living organisms. It is formed during photosynthesis when light energy gathered by chlorophyll is stored in cells. Reactions of UO2 2+ with ATP have been already described [44–46], and Feldman et al. [44] has also postulated a structure of a U-ATP complex. UO2 2+ – ATP complexes are highly stable and the UO2 2+ ion is able to replace metal ions like Mg2+ , Ca2+ , Zn2+ , Cu2+ , Co2+ and Ni2+ in an eventual interaction with ATP in its protonated and/or deprotonated form [46]. The results generated in this study showed that in the pH range of 3 to 4, relevant for AMD environments, more than 90% of the initially added uranium of a 1 × 10−5 mol/L solution was bioaccumulated by Euglena mutabilis cells. At present, it is not possible to relate the bioaccumulation process exclusively to an intra- or extracellular binding mechanism. However, based on the LIFS results and a comparison with uranium reference compounds it can be deduced that under the applied experimental conditions (low pH and environmentally relevant uranium concentrations) aqueous uranium is very likely bioaccumulated either by (1.) (organo)phosphate or by (2.) carboxylic groups of the Euglena cells or by (3.) a combination of the two species.

3.3 Application to uranium contaminated natural environments The results of the above described bioaccumulation experiments with model-solutions containing 1 × 10−5 mol/L uranium opened up the scenario that Euglena cells could be successfully used to clean up real uranium contaminated AMD-waters. Due to the effectiveness to remove uranium at pH-values below pH 4 – a pH range in which uranium does not significantly adsorb to inorganic surfaces under oxic conditions – it gave point to apply Euglena cells in a next logical step to uranium contami-

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nated natural AMD waters. Thus, experiments with Euglena mutabilis cells and sterile filtrated uranium contaminated AMD-water collected from a drainage channel in a gallery of a former underground uranium mining site in Königstein (Saxony, Germany) were carried out in the same way as described for the model bioaccumulation experiments before. The measured pH-value of this natural water was 3.1 ± 0.1 and the uranium concentration was 4.4 × 10−5 mol/L. Two additional bioaccumulation experiments with uranium contaminated AMD-waters were carried out in triplicates. Again, the results were combined and presented as arithmetic means. The results of these two bioaccumulation experiments with Euglena biomasses of 0.60 and 0.74 g dry weight/L, respectively, showed however significant lower uraniumremoval rates of 1.43 and 0.93 mg uranium per g Euglena dry weight. However, these uranium removal rates are still distinctively higher (by a factor of 70 – 320 times) than rates reported [20]. An explanation for the reduced uranium immobilization rates is related to the presence of other components in the natural AMD water sample, which seem to compete for available sorption/binding sites and thereby reduce the uranium bioaccumulation rate by Euglena cells. For example, calcium, magnesium, manganese and zinc, which are present in high concentrations (see Table 2) in the Königstein waters, may also react with Euglena cells and thereby may block potential sorption sites. In addition, organic carbon also sorbs to cell surfaces of phytoplankton cells [47]. This process may also reduce the bioavailability of the metal [21] and thus decrease the effectiveness of the uranium immobilization process on Euglena cells. This study shows that results obtained in laboratory experiments have to be treated with care, since metal immobilization processes by microorganisms occurring in natural environments may be much more complicated and more than one reaction mechanism may compete with each other.

Table 5: Emission maxima of uranium species immobilized by Euglena mutabilis cells in bioaccumulation experiments using water from a drainage channel from the underground mine in Königstein as initial uranium solution. Sample

Drainage water Königstein

Initial solution Euglena cells

Delay [ns] 120, 200 80

1 mv sd (1𝜎) mv sd (1𝜎)

476.9 1.0

Emission bands [nm] 2 3 4 492.6 0.5 494.9 0.5

513.3 0.7 515.9 0.7

536.3 1.2 540.9 1.2

5

6

561.7 0.9 563.3 0.9

590.3 1.6



The Euglena mutabilis cells, after being in contact with the uranium contaminated water from Königstein, were also analyzed by LIFS and the detected positions of the emission maxima are listed in Table 5. The emission bands of the initial solution almost perfectly match with the ones published by Arnold et al. [1] where the uranium speciation in the water samples from the underground mine in Königstein is discussed. As mentioned there the aqueous uranium speciation in the AMD environment of Königstein is dominated in the pH range of 2.5 to 3.0 by the highly mobile aquatic uranium sulfate species UO2 SO4(𝑎𝑞) . The positions of the second, third, fourth and fifth emission maxima (see Table 5) obtained for samples representing bioaccumulated uranium by Euglena mutabilis cells with uranium-containing AMD waters were in good agreement with the uranium emission bands recorded in the previously described bioaccumulation experiments, carried out in Na2 SO4 and NaClO4 media, respectively. From this it can be concluded that the bioaccumulated uranium on or in Euglena cells in contact with natural AMD water is present in the same coordination environment (speciation). A reaction of the aqueous UO2 SO4 uranium species with carboxylic groups and/or with (organo)phosphate groups linked to the Euglena cells is most likely.

4 Conclusions The aim of the present study was to investigate the ability of Euglena mutabilis cells to accumulate uranium from acid solutions. Bioaccumulation experiments were carried out at pH 3 and 4, relevant for uranium contaminated AMD sites. The bioaccumulation experiments were performed in Na2 SO4 media to approximate the sulfate dominated AMD conditions and in NaClO4 as background electrolyte to compare the results with available reference systems. The results of these model experiments showed that Euglena mutabilis cells are able to accumulate significant amounts of uranium from acid solutions. The LIFS investigations indicated that the uranium accumulation is very

likely linked to reactions with carboxylic groups and/or (organo)phosphate groups on or inside the Euglena cells. The bioaccumulation of uranium from contaminated water from an AMD site in Königstein (Saxony, Germany) on the Euglena cells was also studied. In these experiments a smaller uranium-removal rate was observed, which was attributed to the presence of cations and organic carbon in the AMD waters and the resulting competition for available sorption sites. LIFS investigation of these samples on bioaccumulated uranium from uranium contaminated AMD sites showed, however, exactly the same speciation. From this it can be concluded that Euglena cells, which usually live in biofilm communities forming green mats in natural AMD systems, may bioaccumulate uranium from the aqueous phase and eventually become fossilized in sediments similar to observations made by Mann et al. [20]. This study helps to develop an understanding on the bioaccumulation process of uranium by Euglena mutabilis cells on a molecular level. The ability to bind significant amounts of uranium from acid solutions could be advantageous for possible future applications of Euglena mutabilis cells as promising bioaccumulation agent in the treatment and technical cleaning of AMD waters with elevated uranium concentrations, since under these conditions Euglena mutabilis cells are metabolically active and uranium is not sorbing to a significant extent on inorganic surfaces. Acknowledgement: We thank U. Zimmermann and the Wismut GmbH for allowing sampling in the underground mine in Königstein as well as R. Uebe and P. Luz for their assistance in sampling. The Bundesministerium für Bildung und Forschung (BMBF, project number 02NUK002F) and partly the EU project “UMBRELLA” (Using MicroBes for the REgulation of heavy metaL mobiLity at ecosystem and landscape scAle, GA No 226870, project within FP7 topic “Recovery of degraded soil resources”) is thanked for funding. Received July 11, 2013; accepted October 21, 2013.

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